DNA Sequencing 8.2. DNA sample in order to sequence or analyze DNA, more than just a small sample is...

DNA Sequencing

8.2

DNA sample

• in order to sequence or analyze DNA, more than just a small sample is needed

Polymerase Chain Reaction (PCR)

• a direct method of making many copies of a DNA sequence

• each cycle doubles the number of DNA molecules (exponential increase)

• useful in forensics, medical diagnosis and genetic research because a small amount of DNA can be amplified

Taq polymerase

• from Thermus aquaticus (bacterium that lives in hot springs)

• not denatured at the high temperatures needed in PCR

• works optimally at 72ºC

PCR Animations

Look at step-by-step animation of PCR process first:

http://www.dnalc.org/resources/animations/pcr.html

…then this excellent interactive “virtual lab” (one of my favourites):

http://learn.genetics.utah.edu/content/labs/pcr/

Gel Electrophoresis

Gel Electrophoresis

• a method of separating molecules based on size

• DNA migrates through the gel towards a positive electrode

• smaller fragments move faster through the gel, causing separation by size

• DNA fragments can be seen by using a stain such as ethidium bromide

Gel Electrophoresis

Gel Electrophoresis

• Another excellent interactive animation:http://learn.genetics.utah.edu/content/labs/gel/

• Step-by-step slideshow on gel electrophoresis:

http://www.dnalc.org/resources/animations/gelelectrophoresis.html

DNA Sequencing

DNA Sequencing

• chain termination method (Sanger dideoxy method) developed in the 1970s by Frederick Sanger

• Human Genome Project used 2 methods of sequencing, both involving the Sanger method

Deoxy- vs. Dideoxy

Sanger method

Excellent simple narrated animation (the link doesn’t seem to work, but if you cut & paste the address it does…strange):

http://smcg.ccg.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf

McGraw-Hill animation & quiz:http://highered.mcgraw-hill.com/sites/

0072556781/student_view0/chapter15/animation_quiz_1.html

Whole-Genome Shotgun

So you’ve sequenced it…

…what can you do with the data?

• structural genomics

• functional genomics

• bioinformatics

Interesting findings…

• can sequence a gene and then use computer to find similar sequences in the same genome or in other genomes

• have discovered many genes of unknown function

• unexpected degree of similarity between different organisms (e.g., yeast & humans)

$1000 Genome Initiative• currently: about 8 days

and $10 000 to sequence the human genome

• the race is on to be able to sequence the genome faster and for less than $1000

• nanopore sequencing

DNA Microarray

DNA Microarray

• allows scientists to pinpoint the functions of specific genes

• the expression of thousands of genes in a particular cell can be identified simultaneously

Microarray

• microarray or gene chip can hold from 10 to 2.1 million DNA samples (probes)

• could be used to compare a “normal” cell with a cancerous cell

• isolate mRNA from both cells; synthesize labelled cDNA; denature and place on microarray

DNA Microarray

To read the results:

• RED shows gene is expressed in experimental (cancerous) cell

• GREEN show gene is expressed in normal cell

• YELLOW shows gene is expressed in both cells

Microarray Animation

• Excellent step-by-step animation with simplified explanation of process:

http://www.bio.davidson.edu/courses/genomics/chip/chipQ.html